The Ubiquitin-Proteasome System (UPS) is the primary cellular system responsible for the degradation of intracellular proteins. Substrates of the UPS are targeted for degradation by E3 ubiquitin ligases, which recognize degradation signals (degrons) within target proteins, then polyubiquitylate these proteins, targeting them to the proteasome. As such, E3 ubiquitin ligases are key specificity factors within the UPS. However, substrate-linked polyubiquitin chains can subsequently be remodeled by E4 ubiquitin chain-elongating factors which extend polyubiquitin chains, and by deubiquitylases (DUBs), which break these chains down. These competing activities are known to alter the rate of substrate degradation. Recently, the proteasome has been recognized as a major regulatory point in the UPS, containing proteasome-associated factors that exhibit ubiquitin ligase or DUB activity. These associated proteins allow the proteasome to engage in ubiquitin-chain remodeling, giving the proteasome holoenzyme itself the ability to control the degradation rate of substrates. In Saccharomyces cerevisiae, the proteasome-associated Hul5 ubiquitin ligase has been demonstrated to exhibit E4 ubiquitin chain-elongating activity and, remarkably, to regulate the processivity of degradation of engineered proteasomal substrates in vivo. The ability of the proteasome to processively degrade substrates to completion, rather than piecewise, is a fundamental feature of the proteasome. The biochemical basis for how Hul5 enhances proteasomal processivity is currently unclear, as is the full complement of endogenous Hul5 substrates. In the present study, the mechanism by which Hul5 ensures processive proteasomal function will be investigated by reconstitution of the ubiquitylation and degradation reactions using purified proteins. The reaction will be followed using single-molecule technology, providing molecular and temporal resolution not achievable using in vivo or conventional in vitro assays. An existing conditional Ube3c mouse mutant line will be characterized to identify phenotypes resulting from perturbed function of UBE3C, the mammalian ortholog of Hul5. Novel Hul5 and UBE3C substrates will be identified by global proteomics, with an emphasis on tandem mass tag-mass spectrometry (TMT-MS). Identification of endogenous substrates will be an important milestone in the characterization of Hul5/UBE3C activity. Although artificial substrates can serve as powerful biochemical tools for examining enzymatic activity, only through the study of endogenous substrates can mechanistic models be validated and organismal phenotypes explained. The importance of understanding the function of Hul5 and UBE3C, and of identifying natural substrates, is evident from recent studies linking abnormal UBE3C function with malignancy and Autism Spectrum Disorders.